Surface plasmon resonance, SPR, is a sensitive technique to measure optical properties at and close to a surface. SPR setups are used to follow interactions between biomolecules in real time. These interactions are characterized by rate constants such as association and dissociation constants. Moreover, equilibrium constants are measured, as characterization of epitopes of molecules of interest. The analysis involves many different molecules, such as proteins, hormones, DNA, RNA, glycoproteins, receptors, ligands etc. When a molecule binds to the immobilized target, the effective refractive index will increase at the binding spot. This change in refractive index can be detected by use of SPR, where either the resonance angle or the resonance wavelength will change. The SPR condition is given by:
                              k          SP                =                                            2              ⁢              π                        λ                    ⁢                                                                      ɛ                  m                                ·                                  ɛ                  a                                                                              ɛ                  m                                +                                  ɛ                  a                                                                                        (        1        )            
Where, kSP is the propagation constant for the surface plasmon, λ is the wavelength, ∈m is the complex dielectric function, and ∈a is the dielectric function for the ambient media, e.g. biomolecules and liquid. ∈a is equal to an effective refractive index: Na=√{square root over (∈a)}·Na is measured in RIU (refractive index units), which is unitless. For practical reasons small relative RIUs are often measured in mRIU and μRIUs. The resonance condition is fulfilled when the propagation constant of the incident light parallel to the sensor surface matches kSP:
                                                                        2                ⁢                π                            λ                        ⁢                                          n                p                            ·              sin                        ⁢                                                  ⁢            θ                    =                                                    2                ⁢                π                            λ                        ⁢                                                                                ɛ                    m                                    ·                                      ɛ                    a                                                                                        ɛ                    m                                    +                                      ɛ                    a                                                                                      ⁢                                  ⁢                  i          .          e          .                                    (        2        )                                          sin          ⁢                                          ⁢          θ                =                              1                          n              p                                ⁢                                                                      ɛ                  m                                ·                                  ɛ                  a                                                                              ɛ                  m                                +                                  ɛ                  a                                                                                        (        3        )            Where θ is the incident angle, and np is the refractive index of the prism. At the resonance condition, the incident light will be absorbed in the metal film and for that certain wavelength and incident angle the reflected light will be reduced or vanish completely. The dark band that appear is denoted SPR-dip. The width of the dip depends on the wavelength and the metal used. The wavelength for which the resonance occurs is denoted the resonance wavelength.
So far the most common method to follow the interaction is to measure the resonance angle for a fixed wavelength. The resonance angle can be determined by either angular scanning or by projection of the whole angular interval of interest onto a linear detector, using a fan shaped beam. By using a two dimensional detector a line of detection spots can be used. Another method is to use wavelength scanning where the incident angle is held fixed and the wavelengths of interest are varied. The method depends on the wavelength dependence of the surface plasmon supporting metal. Also for this method a line of spots can be detected, using a two-dimensional detector.
There is a demand for detecting many spots, or a detection of a 2-dimensional area, which means that a two-dimensional sensor surface is preferred. This was first described by Yeatman in 1987, and in 1988 by Knoll et al. Because they only had two-dimensional detectors, they measured the intensity changes at the slope of the SPR-resonance dip. Measurement of only the slope of the dip has many drawbacks: limited dynamic range, low sensitivity, sensitivity to offset changes, sensitivity to dip broadening, to mention a few. The dynamic range is limited due to the fact that the SPR dip is sharp and contrast changes can only be detected at the slopes of the SPR-dip. Because an offset change e.g. from a drift at the detector or light source can't be discriminated from a contrast change an offset change will be interpreted as a change of the resonance condition, which is obviously false. The sensitivity will only be high at a small range due to the SPR dip's shape.
Johansen, U.S. Pat. No. 6,862,094, has proposed an apparatus that uses multiple wavelengths to increase the dynamic range and increase sensitivity, precision, accuracy, and throughput. There are several labs and institutions that nowadays use white light in SPR imaging. Wong et al., Tribology International 41 (2008) 356-366, and PhD thesis “Imaging surface plasmon resonance (SPR) photonic sensors”, August 2007, The city Univerity of Hong Kong, uses SPR imaging with white light and use the traditional HSV-model (Hue-Saturation-Value), proposed by Smith, Computer Graphics 1978, 12(3) 12-19, to quantify the position of the SPR-dip. The HSV-method is a cyclic transformation, i.e. it represents a “color whel” with complementary colors. Wong et al. uses the SPR-dip position to make a calibration curve between SPR-position and refractive index. The method has several limitations, because the use of an ordinary camera, a wide spectral RGB-scheme is used, which gives poor sensitivity and is limited to a closed transformation in the visible spectra, and hence a reduced wavelength regime. Because one channel is blue, silver is used which has its plasma frequency above the blue regime, but has very poor chemical properties regarding stability.
By using multi-wavelength SPR imaging, several monochromatic images from the different wavelengths are combined to a pseudo color image, U.S. Pat. No. 6,862,094, Johansen in 2000. The notation color/hue could in this context be any wavelength, wavelength band, hue, or color from below UV (ultraviolet) to above IR and FIR (infrared and far infrared). The pseudo color image is preferably a 2-dimensional image, but can be a line or spot, based on a transformation from two or more monochromatic images, based on the reflectance values from each spot on the monochromatic images, where each spot on the color image has a single value: effective wavelength, dominant wavelength, color, hue or any other arbitrary value that is related to the effective refractive index on the corresponding spot on the sensor surface.